Compositions and methods for regulating angiogenesis

ABSTRACT

In accordance with the present invention, EC progenitors can be used in a method for regulating angiogenesis, i.e., enhancing or inhibiting blood vessel formation, in a selected patient and in some preferred embodiments for targeting specific locations. For example, the EC progenitors can be used to enhance angiogenesis or to deliver an angiogenesis modulator, e.g. anti- or pro-angiogenic agents, respectively to sites of pathologic or utilitarian angiogenesis. Additionally, in another embodiment, EC progenitors can be used to induce reendothelialization of an injured blood vessel, and thus reduce restenosis by indirectly inhibiting smooth muscle cell proliferation.

This application claims priority to U.S. patent application Ser. No.09/228,020, filed Jan. 11, 1999 which claims priority to U.S.application Ser. No. 08/744,882, filed Nov. 8, 1996 now U.S. Pat. No.5,980,887.

FIELD OF THE INVENTION

The invention relates to methods for isolating and using endothelialprogenitor cells and compositions for use in the methods.

BACKGROUND OF THE INVENTION

Blood vessels are the means by which oxygen and nutrients are suppliedto living tissues and waste products removed from living tissue.Angiogenesis is the process by which new blood vessels are formed, asreviewed, for example, by Folkman and Shing, J. Biol. Chem. 267 (16),10931-10934 (1992). Thus angiogenesis is a critical process. It isessential in reproduction, development and wound repair. However,inappropriate angiogenesis can have severe consequences. For example, itis only after many solid tumors are vascularized as a result ofangiogenesis that the tumors begin to grow rapidly and metastasize.Because angiogenesis is so critical to these functions, it must becarefully regulated in order to maintain health. The angiogenesisprocess is believed to begin with the degradation of the basementmembrane by proteases secreted from endothelial cells (EC) activated bymitogens such as vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (bFGF). The cells migrate and proliferate,leading to the formation of solid endothelial cell sprouts into thestromal space, then, vascular loops are formed and capillary tubesdevelop with formation of tight junctions and deposition of new basementmembrane.

In the adults, the proliferation rate of endothelial cells is typicallylow compared to other cell types in the body. The turnover time of thesecells can exceed one thousand days. Physiological exceptions in whichangiogenesis results in rapid proliferation occurs under tightregulation are found in the female reproduction system and during woundhealing.

The rate of angiogenesis involves a change in the local equilibriumbetween positive and negative regulators of the growth of microvessels.Abnormal angiogenesis occurs when the body loses its control ofangiogenesis, resulting in either excessive or insufficient blood vesselgrowth. For instance, conditions such as ulcers, strokes, and heartattacks may result from the absence of angiogenesis normally requiredfor natural healing. On the contrary, excessive blood vesselproliferation may favor tumor growth and spreading, blindness, psoriasisand rheumatoid arthritis.

The therapeutic implications of angiogenic growth factors were firstdescribed by Folkman and colleagues over two decades ago (Folkman, N.Engl. J. Med., 285:1182-1186 (1971)). Thus, there are instances where agreater degree of angiogenesis is desirable—wound and ulcer healing.Recent investigations have established the feasibility of usingrecombinant angiogenic growth factors, such as fibroblast growth factor(FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) andBaffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell growthfactor (ECGF) (Pu, et al., J Surg Res, 54:575-83 (1993)), and morerecently, vascular endothelial growth factor (VEGF) to expedite and/oraugment collateral artery development in animal models of myocardial andhindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) andTakeshita, et al., J Clin Invest, 93:662-70 (1994)).

Conversely, there are also instances, where inhibition of angiogenesisis desirable. For example, many diseases are driven by persistentunregulated angiogenesis. In arthritis, new capillary blood vesselsinvade the joint and destroy cartilage. In diabetes, new capillariesinvade the vitreous, bleed, and cause blindness. Ocularneovascularization is the most common cause of blindness. Tumor growthand metastasis are angiogenesis-dependent. A tumor must continuouslystimulate the growth of new capillary blood vessels for the tumor itselfto grow.

The current treatment of these diseases is inadequate. Agents whichprevent continued angiogenesis, e.g, drugs (TNP-470), monoclonalantibodies and antisense nucleic acids, are currently being tested.However, new agents that inhibit angiogenesis are needed.

Recently, the feasibility of gene therapy for modulating angiogenesishas been demonstrated. For example, promoting angiogenesis in thetreatment of ischemia was demonstrated in a rabbit model and in humanclinical trials with VEGF using a Hydrogel-coated angioplasty balloon asthe gene delivery system. Successful transfer and sustained expressionof the VEGF gene in the vessel wall subsequently augmentedneovascularization in the ischemic limb (Takeshita, et al., LaboratoryInvestigation, 75:487-502 (1996); Isner, et al., Lancet, 348:370(1996)). In addition, it has been demonstrated that direct intramuscularinjection of DNA encoding VEGF into ischemic tissue inducesangiogenesis, providing the ischemic tissue with increased blood vessels(U.S. Ser. No. 08/545,998; Tsurumi et al., Circulation, In Press).

Alternative methods for regulating angiogenesis are still desirable fora number of reasons. For example, it is believed that native endothelialcell (EC) number and/or viability decreases over time. Thus, in certainpatient populations, e.g., the elderly, the resident population of ECsthat is competent to respond to administered angiogenic cytokines may belimited.

Moreover, while agents promoting or inhibiting angiogenesis may beuseful at one location, they may be undesirable at another location.Thus, means to more precisely regulate angiogenesis at a given locationare desirable.

SUMMARY OF THE INVENTION

We have now discovered that by using techniques similar to thoseemployed for HSCs, EC progenitors can be isolated from circulatingblood. In vitro, these cells differentiate into ECs. Indeed, one can usea multipotentiate undifferentiated cell as long as it is still capableof becoming an EC, if one adds appropriate agents to result in itdifferentiating into an EC.

We have also discovered that in vivo, heterologous, homologous, andautologous EC progenitor grafts incorporate into sites of activeangiogenesis or blood vessel injury, i.e. they selectively migrate tosuch locations. This observation was surprising. Accordingly, one cantarget such sites by the present invention.

The present invention provides a method for regulating angiogenesis in aselected patient in need of a change in the rate of angiogenesis at aselected site. The change in angiogenesis necessary may be reduction orenhancement of angiogenesis. This is determined by the disorder to betreated. In accordance with the method of the present invention, aneffective amount of an endothelial progenitor cell or modified versionthereof to accomplish the desired result is administered to the patient.

In order to reduce undesired angiogenesis, for example, in the treatmentof diseases such as rheumatoid arthritis, psoriasis, ocularneovascularization, diabetic retinopathy, neovascular glaucoma,angiogenesis-dependent tumors and tumor metastasis, a modifiedendothelial cell, having been modified to contain a compound thatinhibits angiogenesis, e.g., a cytotoxic compound or angiogenesisinhibitor, can be administered.

To enhance angiogenesis, for example in the treatment of cerebrovascularischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemiccardiomyopathy and myocardial ischemia, endothelial progenitor cells areadministered. To further enhance angiogenesis an endothelial progenitorcell modified to express an endothelial cell mitogen may be used.Additionally, an endothelial cell mitogen or a nucleic acid encoding anendothelial cell mitogen can further be administered.

In another embodiment, the present invention provides methods ofenhancing angiogenesis or treating an injured blood vessel. Inaccordance with these methods, endothelial progenitor cells are isolatedfrom the patient, preferably from peripheral blood, and readministeredto the patient. The patient may also be treated with endothelial cellmitogens to enhance endothelial cell growth. The vessel injury can bethe result of balloon angioplasty, deployment of an endovascular stentor a vascular graft.

The present invention also provides a method of screening for thepresence of ischemic tissue or vascular injury in a patient. The methodinvolves contacting the patient with a labelled EC progenitor anddetecting the labelled cells at the site of the ischemic tissue orvascular injury.

The present invention also includes pharmaceutical products and kit forall the uses contemplated in the methods described herein.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show cell shape and formation. FIG. 1A shows spindle shapedCD34+ attaching cells (AT^(CD34+)) 7 days after plating CD34+mononuclear blood cells (MB^(CD34+)) on fibronectin with standardmedium. Network formation (FIG. 1B) and cord-like structures (FIG. 1C)were observed 48 hours after plating co-culture of MB^(CD34+), labeledwith DiI dye (Molecular Probe), and unlabeled MB^(CD34−) (ratio of1:100) on fibronectin-coated dish. These cords consisted principally ofDiI-labeled MB^(CD34+) derived cells (AT^(CD34+)). Beginning at 12 hoursafter co-culture, MB^(CD34+) derived cells demonstrated multiple foci ofcluster formation (FIGS. 1D, 1E). AT^(CD34+) sprout from the periphery,while round cells remain in the center and detach from the clusterseveral days later. After 5 days, uptake of labeled acetylated lowdensity lipoprotein, acLDL-DiI (Molecular Probe). was seen in AT^(CD34+)cells at the periphery but not at the center of the cluster (FIGS. 1F,1G).

FIG. 2 shows the number of AT^(CD34+) 12 hours and 3 days after singleculture of MB^(CD34+) on plastic alone (CD34±non), collagen coating(CD34⁺/COL), or fibronectin (CD34⁺/FN), and MB^(CD34−) on fibronectin(CD34⁻/FN). AT^(CD34+) yielded significantly higher number of cells at12 hours and 3 days when plated on fibronectin (p<0.05, by ANOVA).

FIG. 3 shows FACS analysis of freshly isolated MB^(CD34+), AT^(CD34+)after 7 days in culture, and HUVECs. Cells were labeled with FITC usingantibodies against CD34, CD31 (Biodesign); Flk-1, Tie-2 (Santa CruzBiotechnology); and CD45. All results were confirmed by triplicateexperiments. Shaded area of each box denotes negative antigen gate;white area denotes positive gate. Numbers indicated for individual gatesdenote percentage of cells determined by comparison with correspondingnegative control labeling.

FIG. 4 shows expression of ecNOS mRNA in MB^(CD34−), MB^(CD34+),AT^(CD34+), human coronary smooth muscle cells (HCSMCs) and HUVECs. DNAwas reverse transcribed from ≈1×10⁶ cells each. Equal aliquots of theresulting DNA were amplified by PCR (40 cycles) with paired primers(sense/antisense: AAG ACA TTT TCG GGC TCA CGC TGC GCA CCC/TGG GGT AGGCAC TTT AGT AGT TCT CCT AAC, SEQ ID NO: 1) to detect ecNOS mRNA. Equalaliquots of the amplified product were analyzed on a 1% agarose gel.Only a single band was observed, corresponding to the expected size (548bp) for ecNOS. Lane 1=MB^(CD34−); Lane 2=MB^(CD34+); Lane 3=AT^(CD34+)after 3 d; Lane 4=AT^(CD34+) after 7 d; Lane 5=HCSMCs; Lane 6=HUVECs.

FIG. 5 is a graph illustrating NO release from AT^(CD34+) was measuredwith an NO-specific polarographic electrode connected to an NO meter(Iso-NO, World Precision Instruments) (Shibuki and Okada, Nature 358,676 (1991).). Calibration of NO electrode was performed daily beforeexperimental protocol according to the following equation:2KNO₂+2KI+2H₂SO₄→2NO+I₂+2H₂O+2K₂SO₄. A standard calibration curve wasobtained by adding graded concentrations of KNO₂ (0-500 nmol/L) tocalibration solution containing KI and H₂SO4. Specificity of theelectrode to NO was previously documented by measurement of NO fromauthentic NO gas (Weyrich, et al, Circ. Res. 75, 692 (1994)). AT^(CD34+)cultured in 6-well plate were washed and then bathed in 5 ml of filteredKrebs-Henseleit solution. Cell plates were kept on a slide warmer (LabLine Instruments) to maintain temperature between 35 and 37° C. For NOmeasurement, sensor probe was inserted vertically into the wells, andthe tip of the electrode remained 2 mm under the surface of thesolution. Measurement of NO, expressed as pmol/10⁵ cells, was performedin a well with incremental doses of VEGF (1, 10, 100 ng/ml) and Ach(0.1, 1, 10 μM). HUVECs and bovine aortic ECs were employed as positivecontrols. For negative control, HCSMCs, NO was not detectable. Allvalues reported represent means of 10 measurements for each group.

FIGS. 6A-6D show co-culture of MB^(CD34+) with HUVECs. Freshly isolatedMB^(CD34+) were labeled with DiI dye and plated on a confluent HUVECmonolayer attached to a fibronectin-coated chamber slide at a density of278 cells/mm² (Nunc). Differentiation of MB^(CD34+) into spindle shapedattaching cells (AT^(CD34+)) (red fluorescence) was observed amongHUVECs within 12 h (FIG. 6A). The AT^(CD34+) number increased onmonolayer for 3 days (FIG. 6B), while meshwork structures were observedin some areas (FIG. 6C). Three days after co-culture, both cells werere-seeded on Matrigel (Becton Dickinson)-coated slides and within 12 hdisclosed capillary network formation, consisting of DiI-labeledAT^(CD34+) and HUVECs (FIG. 6D).

FIG. 7 shows the effect of activated ECs and VEGF on MB^(CD34+)differentiation was investigated by pretreatment of HUVEC with TNF-α (20ng/ml) for 12 hours, and/or incubation of AT^(CD34+)/HUVEC co-culturewith VEGF (50 ng/ml).

FIGS. 8A-8K show sections retrieved from ischemic hindlimb following invivo administration of heterologous (FIGS. 8A-8H), homologous (FIG. 8I),and autologous (FIGS. 8J, 8K) EC progenitors. FIGS. 8A, 8B: Redfluorescence in small inter-muscular artery 6 wks after injection ofDiI-labeled MB^(CD34+). Green fluorescence denotes EC-specific lectinUEA-1. FIG. 8C: DiI (red) and CD31 (green) in capillaries betweenmuscles, photographed through double filter 4 wks after DiI-labeledMB^(CD34+) injection. FIG. 8D: Same capillary structure as in FIG. 8C,showing CD31 expression by MB^(CD34+) which have been incorporated intohost capillary structures expressing CD31. FIGS. 8E, 8F: Immunostaining2 wks after MB^(CD34+) injection shows capillaries comprised ofDiI-labeled MB^(CD34+) derived cells expressing tie-2 receptor (greenfluorescence). Most MB^(CD34+) derived cells are tie-2 positive, and areintegrated with some tie-2 positive native (host) capillary cellsidentified by absence of red fluorescence. FIGS. 8G, 8H: Two wks afterinjection of DiI-labeled MB^(CD34−). Although isolated MB^(CD34−)derived cells (red) can be observed between muscles, but these cells donot express CD31. FIG. 8I: Immunohistochemical β-galactosidase stainingof muscle harvested from ischemic limb of B6,129 mice 4 wks followingadministration of MB^(Flk-1+) isolated from β-galactosidase transgenicmice. Cells overexpressing β-galactosidase (arrows) have beenincorporated into capillaries and small arteries; these cells wereidentified as ECs by anti-CD31 antibody and BS-1 lectin. FIGS. 8J,8K:Sections of muscles harvested from rabbit ischemic hindlimb 4 wks afteradministration of autologous MB^(CD34+). DiI fluorescence (FIG. 8J)indicates localization of MB^(CD34+) derived cells in capillaries seenin phase contrast photomicrograph (FIG. 8K). Each scale bar indicates 50μm.

FIG. 9 is a photograph from a scanning electron microscope showing thatEC progenitors had adhered to the denuded arterial surface and assumed amorphology suggestive of endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

We have now discovered a means to regulate angiogenesis, to promoteangiogenesis in certain subject populations, and to more preciselytarget certain tissues. These methods all involve the use of endothelialcell progenitors. One preferred progenitor cell is an angioblast.

Post-natal neovascularization is believed to result exclusively from theproliferation, migration, and remodeling of fully differentiatedendothelial cells (ECs) derived from pre-existing native blood vessels(Folkman, et al., Circ. Res. 28, 671 (1971); W. Risau, FASEB J. 9, 926(1995)). This adult paradigm, referred to as angiogenesis, contrastswith vasculogenesis, the term applied to formation of embryonic bloodvessels from EC progenitors (Risau, et al., Development 105, 473(1989).).

In contrast to angiogenesis, vasculogenesis typically begins as acluster formation, or blood island, comprised of EC progenitors (e.g.angioblasts) at the periphery and hematopoietic stem cells (HSCs) at thecenter (Flamme and Risau, Development 116, 435 (1992)). In addition tothis intimate and predictable spatial association, such EC progenitorsand HSCs share certain common antigenic determinants, including flk-1,tie-2, and CD-34. Consequently, these progenitor cells have beeninterpreted to derive from a common hypothetical precursor, thehemangioblast (Flamme and Risau, 1992, supra; His, et al., J. Clin.Invest. 97, 591 (1996)).

The demonstration that transplants of HSCs derived from peripheral bloodcan provide sustained hematopoietic recovery constitutes inferentialevidence for circulating stem cells. (Brugger, et al., J. Clin. Oncol.12, 28 (1994)). This observation is now being exploited clinically as analternative to bone marrow transplantation.

We have now discovered that by using techniques similar to thoseemployed for HSCs, EC progenitors can be isolated from circulatingblood. In vitro, these cells differentiate into ECs. Indeed, one can usea multipotentiate undifferentiated cell as long as it is still capableof becoming an EC, if one adds appropriate agents to result in itdifferentiating into an EC.

We have also discovered that in vivo, heterologous, homologous, andautologous EC progenitor grafts incorporate into sites of activeangiogenesis or blood vessel injury, i.e., they selectively migrate tosuch locations. This observation was surprising. Accordingly, one cantarget such sites by the present invention.

In accordance with the present invention, EC progenitors can be used ina method for regulating angiogenesis, i.e., enhancing or inhibitingblood vessel formation, in a selected patient and in some preferredembodiments for targeting specific locations. For example, the ECprogenitors can be used to enhance angiogenesis or to deliver anangiogenesis modulator, e.g. anti- or pro-angiogenic agents,respectively to sites of pathologic or utilitarian angiogenesis.Additionally, in another embodiment, EC progenitors can be used toinduce reendothelialization of an injured blood vessel, and thus reducerestenosis by indirectly inhibiting smooth muscle cell proliferation. Ina still another embodiment, EC progenitors can be used to regeneratetissue, such as cardiac tissue.

In one preferred embodiment the EC cells can be used alone to potentiateangiogeneis in a patient. Some patient populations, typically elderlypatients, may have either a limited number of ECs or a limited number offunctional ECs. Thus, if one desires to promote angiogenesis, forexample, to stimulate vascularization by using a potent angiogenesispromotor such as VEGF, such vascularization can be limited by the lackof ECs. However, by administering the EC progenitors one can potentiatevascularization in those patients.

Accordingly, the present method permits a wide range of strategiesdesigned to modulate angiogenesis such as promoting neovascularizationof ischemic tissues (Isner, et al, Lancet 348, 370 (1996)). EC mitogenssuch as VEGF and bFGF, for example, have been employed to stimulatenative ECs to proliferate, migrate, remodel and thereby form new sproutsfrom parent vessels (D′Amore and Thompson, Annu. Rev. Physiol. 49, 453(1987)). A potentially limiting factor in such therapeutic paradigms isthe resident population of ECs that is competent to respond toadministered angiogenic cytokines. The finding that NO productiondeclines as a function of age (Tschudi, et al, J. Clin. Invest. 98, 899(1996)) may indicate a reduction in EC number and/or viability thatcould be addressed by autologous EC grafting. The success demonstratedto date with autologous grafts of HSCs derived from peripheral blood(Brugger, et al., 1995, supra) Kessinger and Armitage, Blood 77, 211(1991); Sheridan, et al., Lancet 339, 640 (1992); Shpall, et al., J.Clin. Oncol. 12, 28 (1994)) supports the clinical feasibility of a“supply side” approach to therapeutic angiogenesis. The in vivo data setforth herein indicate that autologous EC transplants are feasible, andthe in vitro experiments indicate that EC progenitors(MB^(CD34+)-derived ECs) can be easily manipulated and expanded ex vivo.

Our discovery that these EC progenitors home to foci of angiogenesismakes these cells useful as autologous vectors for gene therapy anddiagnosis of ischemia or vascular injury. For example, these cells canbe utilized to inhibit as well as augment angiogenesis. Foranti-neoplastic therapies, for example, EC progenitors can betransfected with or coupled to cytotoxic agents, cytokines orco-stimulatory molecules to stimulate an immune reaction, otheranti-tumor drugs or angiogenesis inhibitors. For treatment of regionalischemia, angiogenesis could be amplified by prior transfection of ECprogenitors to achieve constitutive expression of angiogenic cytokinesand/or selected matrix proteins (Sato, et al, Exp. Cell Res. 204, 223(1993); Pepper, et al., Biochem Biophys Res Comm 181, 902 (1991);Senger, et al, Am. J. Pathol. 149, 293 (1996)). In addition, the ECprogenitors may be labelled, e.g., radiolabelled, administered to apatient and used in the detection of ischemic tissue or vascular injury.

EC progenitors may be obtained from human mononuclear cells obtainedfrom peripheral blood or bone marrow of the patient before treatment. ECprogenitors may also be obtained from heterologous or autologousumbilical cord blood. Peripheral blood is preferred due to convenience.The leukocyte fraction of peripheral blood is most preferred. ECprogenitors may be isolated using antibodies that recognize ECprogenitor specific antigens on immature human hematopoietic progenitorcells (HSCs). For example, CD34 is commonly shared by EC progenitor andHSCs. CD34 is expressed by all HSCs but is lost by hematopoietic cellsas they differentiate (Civin, et al, J Immunol 133, 157 (1984); Katz, etal., Leuk. Res. 9, 191 (1985); Andrews, et al., Blood 67, 842 (1986)).It is also expressed by many, including most activated, ECs in the adult(Fina, et al, Blood 75, 17 (1990); Soligo, et al, Leukemia 5, 1026(1991); Ito, et al., Lab. Invest. 72, 532 (1995)). Flk-1, a receptor forvascular endothelial growth factor (VEGF) (deVries, et al, Science 255,989 (1992); Terman, et al, Biochem. Biophys. Res. Commun. 187, 1579(1992); Shalaby, et al, Nature 376, 62 (1995)), is also expressed byboth early HSCs and ECs, but ceases to be expressed in the course ofhematopoietic differentiation (Matthews, et al, Proc. Natl. Acad. Sci.USA. 88, 9026 (1991); Millauer, et al, Cell 72, 835 (1993); Yamaguchi,et al., Development 118, 489 (1993)).

To obtain the EC progenitors from peripheral blood about 5 ml to about500 ml of blood is taken from the patient. Preferably, about 50 ml toabout 200 ml of blood is taken.

EC progenitors can be expanded in vivo by administration of recruitmentgrowth factors, e.g., GM-CSF and IL-3, to the patient prior to removingthe progenitor cells.

Methods for obtaining and using hematopoietic progenitor cells inautologous transplantation are disclosed in U.S. Pat. No. 5,199,942, thedisclosure of which is incorporated by reference.

Once the progenitor cells are obtained by a particular separationtechnique, they may be administered to a selected patient to treat anumber of conditions including, but not limited to: unregulatedangiogenesis, blood vessel injury, ischemia, myocardial infarction,myocardial injury, restoration of left ventricular (LV) function,vascular occlusion or stenosis, peripheral vascular disease, sickle cellanemia, thalassemia, and the various conditions discussed suprarequiring inducing or enhancing angiogenesis. Additionally, theprogenitor cells may be used to vascularize grafts of transplantedtissue (e.g., such as skin grafts), to regenerate cells, to formbioprostheses (see, e.g., as discussed in U.S. Pat. No. 6,375,680), andin surgical procedures such as CABG. The progenitor cells may also beused to deliver inhibitory agents to tissues and cells in need, todecrease angiogeneis where desirable, e.g., to treat conditions such astumor growth, diabetic retinopathy, rheumatoid arthritis, and chronicinflammatory diseases (see, U.S. Pat. No. 5,318,957; Yancopoulos, et al.Cell 93: 661-4 (1998); Folkman, et al., Cell 87: 1153-5 (1996); andHanahan, et al., Cell 86: 353-64 (1996)).

The cells may be stored under cryogenic conditions. Optionally, thecells may be expanded ex vivo using, for example, the method disclosedby U.S. Pat. No. 5,541,103, the disclosure of which is incorporated byreference.

Preferably, the progenitor cells obtained from the patient arereadministered. Generally, from about 10⁶ to about 10¹⁸ progenitor cellsare administered to the patient.

The progenitor cells are administered to the patient by any suitablemeans, including, for example, intravenous infusion, bolus injection,and site-directed delivery via a catheter (e.g., such as an endocardialdelivery catheter), stent, syringe, transthoracic drug delivery device(U.S. Pat. No. 6,517,527) or other medical access device or implantabledevice. As discussed above, progenitor cells may be administered withmitogens (e.g., such as VEGF), other agents for modulating angiogenesis(e.g., proteins, peptides, nucleic acids, drugs, small molecules and thelike), and even other types of progenitor cells (see, e.g., U.S. PatentPublication Ser. No. 20020142457). Mitogens and agents may be deliveredthrough the lumen of a medical access device and/or may coat at least aportion of the inner and/or outer walls of such a device.

In certain aspects, it is desirable to deliver an endothelial progenitorto specific cells in need of angiogenesis within a tissue. In oneaspect, endothelial progenitor cells are delivered to zones ofhibernating myocardium or to sites of myocardial infarction to preserveLV function.

For example, cells in need of angiogenesis may be identified byendomyocardial or electromechanical mapping (EMM) of cardiac tissueusing, for example, the NOGA system (Biosense-Webster) of catheter-basedmapping and navigation (see, Kawamoto, et al., Circulation 107(3), 461-8(2003), and references cited therein). The editing of the raw data maybe performed by a suitable system such as the NOGA system computer.

After mapping, a suitable medical access device capable of deliveringone or more endothelial progenitor cells (a “delivery device”) (e.g.,such as a percutaneous injection catheter) is positioned in proximity tocell(s) in need (such as at a zone of ischemia) identified by mapping.An intracardiac electrogram may be obtained to detect transientmyocardial injury and/or premature ventricular contractions as evidenceof penetration of at least a portion of the delivery device into themyocardium. After positioning, progenitor cells are delivered (e.g., byinjection) and the process can be repeated again as necessary to deliveradditional progenitors to additional cell(s) in need. ECG monitoring andcreatine kinase monitoring and other assays may be used to monitor thegeneral cardiac health of the patient. SPECT Myocardial Perfusionstudies may be performed, as known in the art, to monitor angiogenesis.

Depending on the use of the progenitor cells, various genetic materialmay be delivered to the cell. The genetic material that is delivered tothe EC progenitors may be genes, for example, those that encode avariety of proteins including anticancer agents. Such genes includethose encoding various hormones, growth factors, enzymes, cytokines,receptors, MHC molecules, telomerase, and the like. The term “genes”includes nucleic acid sequences both exogenous and endogenous to cellsinto which a virus vector, for example, a pox virus such as swine poxcontaining the human TNF gene may be introduced.

Additionally, it is of interest to use genes encoding polypeptides forsecretion from the EC progenitors so as to provide for a systemic effectby the protein encoded by the gene. Specific genes of interest includethose encoding TNF, TGF-α, TGF-β, hemoglobin, interleukin-1,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin-12 etc., GM-CSF, G-CSF,M-CSF, human growth factor, co-stimulatory factor B7, insulin, factorVIII, factor IX, PDGF, EGF, NGF, IL-ira, EPO, β-globin, EC mitogens andthe like, as well as biologically active muteins of these proteins. Thegene may further encode a product that regulates expression of anothergene product or blocks one or more steps in a biological pathway. Inaddition, the gene may encode a toxin fused to a polypeptide, e.g., areceptor ligand, or an antibody that directs the toxin to a target, suchas a tumor cell. Similarly, the gene may encode a therapeutic proteinfused to a targeting polypeptide, to deliver a therapeutic effect to adiseased tissue or organ.

The cells can also be used to deliver genes to enhance the ability ofthe immune system to fight a particular disease or tumor. For example,the cells can be used to deliver one or more cytokines (e.g., IL-2) toboost the immune system and/or one or more antigens.

These cells may also be used to selectively administer drugs, such as anantiangiogenesis compound such as O-chloroacetyl carbamoyl fumagillol(TNP-470). Preferably the drug would be incorporated into the cell in avehicle such as a liposome, a timed released capsule, etc. The ECprogenitor would then selectively hone in on a site of activeangiogenesis such as a rapidly growing tumor where the compound would bereleased. By this method, one can reduce undesired side effects at otherlocations.

In one embodiment, the present invention may be used to enhance bloodvessel formation in ischemic tissue, i.e., a tissue having a deficiencyin blood as the result of an ischemic disease. Such tissues can include,for example, muscle, brain, kidney and lung. Ischemic diseases include,for example, cerebrovascular ischemia, renal ischemia, pulmonaryischemia, limb ischemia, ischemic cardiomyopathy and myocardialischemia.

If it is desirable to further enhance angiogenesis, endothelial cellmitogens may also be administered to the patient in conjunction with, orsubsequent to, the administration of the EC progenitor cells.Endothelial cell mitogens can be administered directly, e.g.,intra-arterially, intramuscularly, or intravenously, or nucleic acidencoding the mitogen may be used. See, Baffour, et al., supra (bFGF);Pu, et al, Circulation, 88:208-215 (1993) (aFGF); Yanagisawa-Miwa, etal., supra (bFGF); Ferrara, et al., Biochem. Biophys. Res. Commun.,161:851-855 (1989) (VEGF); (Takeshita, et al., Circulation, 90:228-234(1994)).

The nucleic acid encoding the EC mitogen can be administered to a bloodvessel perfusing the ischemic tissue or to a site of vascular injury viaa catheter, for example, a hydrogel catheter, as described by U.S. Ser.No. 08/675,523, the disclosure of which is herein incorporated byreference.

The nucleic acid also can be delivered by injection directly into theischemic tissue using the method described in U.S. Ser. No. 08/545,998.

As used herein the term “endothelial cell mitogen” means any protein,polypeptide, mutein or portion that is capable of, directly orindirectly, inducing endothelial cell growth. Such proteins include, forexample, acidic and basic fibroblast growth factors (aFGF and bFGF),vascular endothelial growth factor (VEGF), epidermal growth factor(EGF), transforming growth factor α and β (TGF-α and TFG-β),platelet-derived endothelial growth factor (PD-ECGF), platelet-derivedgrowth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growthfactor (HGF), insulin like growth factor (IGF), erythropoietin, colonystimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophageCSF (GM-CSF) and nitric oxidesynthase (NOS). See, Klagsbrun, et al.,Annu. Rev. Physiol., 53, 217-239 (1991); Folkmnan, et al., J. Biol.Chem., 267, 10931-10934 (1992) and Symes, et al., Current Opinion inLipidology, 5, 305-312 (1994). Muteins or fragments of a mitogen may beused as long as they induce or promote EC cell growth.

Preferably, the endothelial cell mitogen contains a secretory signalsequence that facilitates secretion of the protein. Proteins havingnative signal sequences, e.g., VEGF, are preferred. Proteins that do nothave native signal sequences, e.g., bFGF, can be modified to containsuch sequences using routine genetic manipulation techniques. See, Nabelet al., Nature, 362, 844 (1993).

The nucleotide sequence of numerous endothelial cell mitogens, arereadily available through a number of computer databases, for example,GenBank, EMBL and Swiss-Prot. Using this information, a DNA segmentencoding the desired may be chemically synthesized or, alternatively,such a DNA segment may be obtained using routine procedures in the art,e.g., PCR amplification. A DNA encoding VEGF is disclosed in U.S. Pat.No. 5,332,671, the disclosure of which is herein incorporated byreference.

In certain situations, it may be desirable to use nucleic acids encodingtwo or more different proteins in order optimize the therapeuticoutcome. For example, DNA encoding two proteins, e.g., VEGF and bFGF,can be used, and provides an improvement over the use of bFGF alone. Oran angiogenic factor can be combined with other genes or their encodedgene products to enhance the activity of targeted cells, whilesimultaneously inducing angiogenesis, including, for example, nitricoxide synthase, L-arginine, fibronectin, urokinase, plasminogenactivator and heparin.

The term “effective amount” means a sufficient amount of compound, e.g.nucleic acid delivered to produce an adequate level of the endothelialcell mitogen, i.e., levels capable of inducing endothelial cell growthand/or inducing angiogenesis. Thus, the important aspect is the level ofmitogen expressed. Accordingly, one can use multiple transcripts or onecan have the gene under the control of a promoter that will result inhigh levels of expression. In an alternative embodiment, the gene wouldbe under the control of a factor that results in extremely high levelsof expression, e.g., tat and the corresponding tar element.

The EC progenitors may also be modified ex vivo such that the cellsinhibit angiogenesis. This can be accomplished, for example, byintroducing DNA encoding angiogenesis inhibiting agents to the cells,using for example the gene transfer techniques mentioned herein.Angiogenesis inhibiting agents include, for example, proteins such asthrombospondin (Dameron, et al., Science 265, 1582-1584 (1994)),angiostatin (O'Reilly, et al., Cell 79, 315-328 (1994), IFN-alpha(Folkman, J. Nature Med. 1:27-31 (1995)), transforming growth factorbeta, tumor necrosis factor alpha, human platelet factor 4 (PF4);substances which suppress cell migration, such as proteinase inhibitorswhich inhibit proteases which may be necessary for penetration of thebasement membrane, in particular, tissue inhibitors of metalloproteinaseTIMP-1 and TIMP-2; and other proteins such as protamine which hasdemonstrated angiostatic properties, decoy receptors, drugs such asanalogues of the angioinhibin fumagillin, e.g., TNP-470 (Ingber, et al.,Nature 348, 555-557 (1990), antibodies or antisense nucleic acid againstangiogenic cytokines such as VEGF. Alternatively, the cells may becoupled to such angiogenesis inhibiting agent.

If the angiogenesis is associated with neoplastic growth the ECprogenitor cell may also be transfected with nucleic acid encoding, orcoupled to, anti-tumor agents or agents that enhance the immune system.Such agents include, for example, TNF, cytokines such as interleukin(IL) (e.g., IL-2, IL-4, IL-10, IL-12), interferons (IFN) (e.g., IFN-γ)and co-stimulatory factor (e.g., B7). Preferably, one would use amultivalent vector to deliver, for example, both TNF and IL-2simultaneously.

The nucleic acids are introduced into the EC progenitor by any methodwhich will result in the uptake and expression of the nucleic acid bythe cells. These can include vectors, liposomes, naked DNA,adjuvant-assisted DNA, catheters, gene gun, etc. Vectors includechemical conjugates such as described in WO 93/04701, which hastargeting moiety (e.g. a ligand to a cellular surface receptor), and anucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNAor RNA viral vector), fusion proteins such as described in WO 95/22618,which is a fusion protein containing a target moiety (e.g. an antibodyspecific for a target cell) and a nucleic acid binding moiety (e.g. aprotamine), plasmids, phage, etc. The vectors can be chromosomal,non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia virusesand HIV-based viruses. One preferred HIV-based viral vector comprises atleast two vectors wherein the gag and pol genes are from an HIV genomeand the env gene is from another virus. DNA viral vectors are preferred.These vectors include pox vectors such as orthopox or avipox vectors,herpesvirus vectors such as a herpes simplex I virus (HSV) vector[Geller, et al., J. Neurochem, 64, 487 (1995); Lim, F., et al., in DNACloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, OxfordEngland) (1995); Geller, et al., Proc Natl. Acad. Sci.: USA. 90, 7603(1993); Geller, et al., Proc Natl. Acad. Sci USA: 87, 1149 (1990)],Adenovirus Vectors [LaSalle, et al., Science, 259, 988 (1993); Davidson,et al., Nat. Genet 3, 219 (1993); Yang, et al., J. Virol. 69, 2004(1995)] and Adeno-associated Virus Vectors [Kaplitt, M. G., et al., Nat.Genet. 8, 148 (1994)].

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g.,infection, transfection, transduction or transformation. Examples ofmodes of gene transfer include e.g., naked DNA, CaPO₄ precipitation,DEAE dextran, electroporation, protoplast fusion, lipofecton, cellmicroinjection, viral vectors and use of the “gene gun”.

To simplify the manipulation and handling of the nucleic acid encodingthe protein, the nucleic acid is preferably inserted into a cassettewhere it is operably linked to a promoter. The promoter must be capableof driving expression of the protein in cells of the desired targettissue. The selection of appropriate promoters can readily beaccomplished. Preferably, one would use a high expression promoter. Anexample of a suitable promoter is the 763-base-pair cytomegalovirus(CMV) promoter. The Rous sarcoma virus (RSV) (Davis, et al., Hum. GeneTher. 4, 151 (1993)) and MMT promoters may also be used. Certainproteins can expressed using their native promoter. Other elements thatcan enhance expression can also be included such as an enhancer or asystem that results in high levels of expression such as a tat gene andtar element. This cassette can then be inserted into a vector, e.g., aplasmid vector such as pUC118, pBR322, or other known plasmid vectors,that includes, for example, an E. coli origin of replication. See,Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, (1989). The plasmid vector may also include aselectable marker such as the β-lactamase gene for ampicillinresistance, provided that the marker polypeptide does not adverselyeffect the metabolism of the organism being treated. The cassette canalso be bound to a nucleic acid binding moiety in a synthetic deliverysystem, such as the system disclosed in WO 95/22618.

If desired, the preselected compound, e.g. a nucleic acid such as DNAmay also be used with a microdelivery vehicle such as cationic liposomesand adenoviral vectors. For a review of the procedures for liposomepreparation, targeting and delivery of contents, see Mannino andGould-Fogerite, BioTechniques, 6, 682 (1988). See also, Felgner andHolm, Bethesda Res. Lab. Focus 11(2), 21 (1989) and Maurer, R. A.,Bethesda Res. Lab. Focus 11 (2), 25 (1989).

Replication-defective recombinant adenoviral vectors, can be produced inaccordance with known techniques. See, Quantin, et al., Proc. Natl.Acad. Sci. USA 89, 2581-2584 (1992); Stratford-Perricadet, et al., J.Clin. Invest. 90, 626-630 (1992); and Rosenfeld, et al., Cell, 68,143-155 (1992).

The effective dose of the nucleic acid will be a function of theparticular expressed protein, the target tissue, the patient and his orher clinical condition. Effective amount of DNA are between about 1 and4000 μg, more preferably about 1000 and 2000, most preferably betweenabout 2000 and 4000.

Alternatively, the EC progenitors may be used to inhibit angiogenesisand/or neoplastic growth by delivering to the site of angiogenesis acytotoxic moiety coupled to the cell. The cytotoxic moiety may be acytotoxic drug or an enzymatically active toxin of bacterial, fungal orplant origin, or an enzymatically active polypeptide chain or fragment(“A chain”) of such a toxin. Enzymatically active toxins and fragmentsthereof are preferred and are exemplified by diphtheria toxin Afragment, non-binding active fragments of diphtheria toxin, exotoxin A(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alphasarcin, certain Aleurites fordii proteins, certain Dianthinproteins, Phytolacca americana proteins (PAP, PAPII and PAP-S),Momordica charantia inhibitor, curcin, crotin, Saponaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin,Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAP are preferred.

Conjugates of the EC progenitors and such cytotoxic moieties may be madeusing a variety of coupling agents. Examples of such reagents areN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters such as dimethyladeipimidate HCI, active esters such as disuccinimidyl suberate,aldehydes such as glutaradehyde, bis-azido compounds such asbis(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene2,6-diisocyante, and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene.

The enzymatically active polypeptide of the toxins may be recombinantlyproduced. Recombinantly produced ricin toxin A chain (rRTA) may beproduced in accordance with the methods disclosed in PCT W085/03508.Recombinantly produced diphtheria toxin A chain and non-binding activefragments thereof are also described in PCT W085/03508.

The methods of the present invention may be used to treat blood vesselinjuries that result in denuding of the endothelial lining of the vesselwall. For example, primary angioplasty is becoming widely used for thetreatment of acute myocardial infarction. In addition, endovascularstents are becoming widely used as an adjunct to balloon angioplasty.Stents are useful for rescuing a sub-optimal primary result as well asfor diminishing restenosis. To date, however, the liability of theendovascular prosthesis has been its susceptibility to thromboticocclusion in approximately 3% of patients with arteries 3.3 mm orlarger. If patients undergo stent deployment in arteries smaller thanthis the incidence of sub-acute thrombosis is even higher. Sub-acutethrombosis is currently prevented only by the aggressive use ofanticoagulation. The combination of vascular intervention and intenseanticoagulation creates significant risks with regard to peripheralvascular trauma at the time of the stent/angioplasty procedure.Acceleration of reendothelialization by administration of EC progenitorsto a patient undergoing, or subsequent to, angioplasty and/or stentdeployment can stabilize an unstable plaque and prevent re-occlusion.

The method of the present invention may be used in conjunction with themethod for the treatment of vascular injury disclosed in PCT/US96/15813.

In addition, the methods of the present invention may be used toaccelerate the healing of graft tissue, e.g., vascular grafts.

In a further aspect, the methods of the invention may be used tostimulate tissue regeneration and in particular, cardiac regeneration.For example, autologous progenitor cells are provided to patients inneed of cardiac tissue regeneration (e.g., such as patients withcoronary artery disease (CAD), myocardial infarctions, etc.), andcontacted with one or more cardiomyocytes, thereby stimulating thedifferentiation of the progenitor cells to form additionalcardiomyocytes. Conversion of progenitor cells to cardiomyocytes can bemonitored by evaluating the expression of appropriate markers (e.g.,cardiac troponin I, atrial natriuretic peptide, α-sarcomeric actinin,MEF-2, and the like) and/or the uptake of diacylated LDL (sinceendothelial progenitor cells can take up diacylated LDL butcardiomyocytes cannot), measuring calcium transits, etc. Contacting mayoccur in vivo or ex vivo. Endothelial progenitor cells for stimulatingcardiac regeneration may be delivered to cardiac tissue by injection,preferably, in situ (e.g., via an endocardial delivery catheter or othersuitable medical access device).

Endothelial progenitors can be selected for any of the above-describedmethods by obtaining mononuclear cells (e.g., from blood or bonemarrow), and selecting for one or more, two or more, or three or more ofthe following markers: CD34⁺, Flk-1⁺, tie-2⁺′, CD31⁺, selectin,endothelial marker proteins (such as von Willebrand factor, vascularendothelial cadherin, and endothelial nitric oxide synthase), or one ormore of the following properties: uptake of diacetylated LDL(DiI-acLDL), and lectin binding.

In one preferred aspect, the markers: CD34⁺, Flk-1⁺, tie-2⁺′ are used.

The present invention also includes pharmaceutical products for all theuses contemplated in the methods described herein. For example, there isa pharmaceutical product, comprising nucleic acid encoding anendothelial cell mitogen and EC progenitors, in a physiologicallyacceptable administrable form.

The present invention further includes a kit for the in vivo systemicintroduction of an EC progenitor and an endothelial cell mitogen ornucleic acid encoding the same into a patient. Such a kit includes acarrier solution, nucleic acid or mitogen, and a means of delivery,e.g., a catheter or syringe. The kit may also include instructions forthe administration of the preparation.

EXAMPLES

The present invention is further illustrated by the following examples.These examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

Example 1 Method and Materials

Human peripheral blood was obtained using a 20 gauge intravenouscatheter, discarding the first 3 ml. Leukocyte fraction of blood wasobtained by Ficoll density gradient centrifugation and plated on plastictissue culture for 1 hr to avoid contamination by differentiatedadhesive cells.

Fluorescent activated cell sorting (FACS) was carried out with >1×10⁶CD34 positive and negative mononuclear blood cells (MB^(CD34+),MB^(CD34−)) Cells were analyzed with Becton-Dickinson FACS sorter andthe lysis II analysis program using antibodies to CD34 (Biodesign).

M-199 medium with 20% FBS and bovine brain extract (Clonetics) was usedas standard medium for all cell culture experiments.

C57BL/6Jx129/SV background male mice (Hirlan), 3 mo old and 20-30 g,were used in these experiments (n=24). Animals were anesthetized with160 mg/kg intraperitoneally of pentobarbital. The proximal end of onefemoral artery and distal portion of the corresponding saphenous arterywere ligated, following which the artery, as well as all side-branches,were dissected free and excised. (All protocols were approved by St.Elizabeth's Institutional Animal Care and Use Committee.)

New Zealand White rabbits (3.8-4.2 kg, n=4, Pine Acre Rabbitry) wereanesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0.8mg/kg) following premedication with xylazine (2 mg/kg). After alongitudinal incision, the femoral artery was dissected free along itsentire length; all branches of the femoral artery were also dissectedfree. After ligating the popliteal and saphenous arteries distally, theexternal iliac artery proximally and all femoral arterial branches, thefemoral artery was completely excised (Takeshita, et al, J. Clin.Invest. 93, 662 (1994); Pu, et al, Circulation 88, 208 (1993); Baffour,et al, J. Vasc. Surg. 16, 181 (1992)).

Isolation and Analysis

CD34 positive mononuclear blood cells (MB^(CD34+)) were isolated fromperipheral blood by CD34 antibody-coated magnetic beads (Dynal) asdescribed above.

FACS analysis indicated that 15.9±3.3% of selected cells versus <0.1% ofthe remaining cells expressed CD34. Depleted (MB^(CD34+)) cells wereused as controls. Flk-1 antibody was used for magnetic bead selection ofFlk-1 positive mononuclear blood cells (MB^(Flk1+)).

MB^(CD34+) and MB^(CD34−) were plated separately in standard medium ontissue culture plastic, collagen type I, or fibronectin. When plated ontissue culture plastic or collagen at a density of 1×10³/mm², a limitednumber of MB^(CD34+) attached, and became spindle shaped andproliferated for 4 wks. A subset of MB^(CD34+) plated on fibronectinpromptly attached and became spindle shaped within 3 days (FIG. 1A); thenumber of attaching cells (AT^(CD34+)) in culture increased with time(FIG. 2). Attached cells were observed only sporadically among culturesof MB^(CD34−), including cells followed for up to 4 wks onfibronectin-coated plates.

To confirm that spindle-shaped cells were derived from CD34 positivecells, MB^(CD34+) were labeled with the fluorescent dye, DiI, andco-plated with unlabeled MB^(CD34−) on fibronectin at an overall densityof 5×10³/mm²; ratio of the two cell types was identical to that of theoriginal mononuclear cell population (1% MB^(CD34+), 99% MB^(CD34−)).Seven days later, DiI-labeled cells derived from MB^(CD34+), initiallyaccounting for only 1% of blood cells, accounted for 60.3±4.7% of totalattaching cells analyzed by FACS. Co-incubation with MB^(CD34−)increased proliferation to >10× MB^(CD34+) plated alone at a celldensity of 5×10/mm² cell (d 3=131.3±26.8 vs 9.7±3.5/mm²).MB^(CD34+)/MB^(CD34−) co-cultures also enhanced MB^(CD34+)differentiation, including formation of cellular networks and tube-likestructures on fibronectin-coated plates (FIGS. 1B, C). These structuresconsisted principally of DiI-labeled MB^(CD34+) derived cells (FIG. 1C).Moreover, within 12 h of co-culture, multiple cluster formations wereobserved (FIG. 1D), consisting principally of DiI-labeled MB^(CD34+)derived cells (FIG. 1E). These clusters were comprised of round cellscentrally, and sprouts of spindle-shaped cells at the periphery. Theappearance and organization of these clusters resembled that of bloodisland-like cell clusters observed in dissociated quail epiblastculture, which induced ECs and gave rise to vascular structures in vitro(Flamme and Risau, 1982, supra). AT^(CD34+) at the cluster peripherywere shown to take up DiI-labeled acetylated LDL, characteristic of EClineage (Voyta, et al., J. Cell Biol. 99, 2034 (1984)), whereas theround cells comprising the center of cluster did not (FIGS. 1F,G); thelatter detached from the cluster several days later. Similar findingswere observed in the experiments using MB^(Flk1+).

Expression of Leukocyte and EC Markers

To further evaluate progression of MB^(CD34+) to an EC-like phenotype,cells were assayed for expression of leukocyte and EC markers. Freshlyisolated MB^(CD34+) versus AT^(CD34+) cultured at densities of 1×10³cell/mm² for 7 days were incubated with fluorescent-labeled antibodiesand analyzed by FACS (FIG. 3). Leukocyte common antigen, CD45, wasidentified on 94.1% of freshly isolated cells, but was essentially lostby 7 d in culture (FIG. 3). Augmented expression of UEA-1, CD34, CD31,Flk-1, Tie-2 and E-selectin—all denoting EC lineage (Millauer, et al,Cell 72, 835 (1993); Yamaguchi, et al., Development 118, 489 (1993);Miettinen, et al., Am. J. Clin. Pathol. 79, 32 (1983); Jaffe, et al., J.Clin. Invest. 52, 2745 (1973); Newman, et al, Science 247, 1219 (1990);Vecchi, et al, Eur. J. Cell Biol. 63, 247 (1994); TSato, et al, Nature376, 70 (1995); Schnurch and Risau, Development 119, 957 (1993);Bevilacqua, Annu. Rev. Immuno. 11, 767 (1993))—was detected amongAT^(CD34+) after 7 days in culture, compared to freshly isolatedMB^(CD34+). CD68 expression, suggesting monocyte/macrophage lineage, waslimited to 6.0±2.4% cells.

Expression of Factor VIII, UEA-1, CD31, ecNOS, and E-selectin was alsodocumented by immunohistochemistry for AT^(CD34+) after 7 days culture(data not shown). After 3, 7, and 14 days in culture, more than 80%AT^(CD34+) took up DiI-labeled acLDL (Voyta, 1984, supra).

ECs uniquely express endothelial constitutive nitric oxide synthase(ecNOS). Accordingly, MB^(CD34+), MB^(CD34−) and AT^(CD34+) wereinvestigated for expression of ecNOS by RT-PCR (Janssens, et al., J.Biol. Chem. 267, 14519 (1992); Lamos, et al., Proc. Natl. Acad. Sci. USA89, 6348 (1992)). ecNOS mRNA was not detectable among MB^(CD34−) and waspresent at very low levels in freshly isolated MB^(CD34+)+(FIG. 4). InAT^(CD34+) cultured for 7 d, however, ecNOS mRNA was markedly increased(FIG. 5). Functional evidence of ecNOS protein in AT^(CD34+) wasdocumented by measurement of nitric oxide in response to theEC-dependent agonist, acetylcholine (Ach), and the EC-specific mitogen,vascular endothelial growth factor (VEGF) (FIG. 5); the latterparenthetically constitutes evidence for a functional Flk-1 receptor aswell among AT^(CD34+).

Cell-Cell Interaction

Cell-cell interaction is considered to play a decisive role in cellsignaling, differentiation, and proliferation during hematopoiesis(Torok-Storb, Blood 72, 373 (1988); N. Dainiak, Blood 78, 264 (1991))and angiogenesis (Folkman and Klagsbrun, Science 235, 442 (1987); Hynes,Cell 48, 549 (1987); Brooks, et al., Science 264, 569 (1994);Friedlander, et al, Science 270, 1500 (1995)). To study the impact ofMB^(CD34+) interaction with mature ECs on the differentiation ofMB^(CD34−) into an EC-like phenotype, DiI-labeled MB^(CD34+) were platedon a confluent HUVEC monolayer. Adherent, labeled cells were foundthroughout the culture within 12 h (FIG. 6A), and increased in numberfor up to 3 d (FIG. 6B). When incubated with 50 ng/ml VEGF and 10 ng/mlbFGF, a meshwork of cord-like structures comprised of both DiI-labeledand unlabeled cells could be seen within 3 d after co-culture (FIG. 6C).Both cell types were then re-seeded on Matrigel (Becton Dickinson)coated slides and within 12 h demonstrated formation of capillarynetworks comprised of DiI-labeled MB^(CD34+) derived cells and HUVECs(FIG. 6D). To facilitate cell-cell interaction, HUVECs were pre-treatedwith TNF-α (Simmons, et al, Blood 80, 388 (1992); Liesveld, et al.,Leukemia 8, 2111 (1994)), resulting in increased numbers of AT^(CD34+)(FIG. 6E); synergistic augmentation was observed upon co-incubation withVEGF. Identically treated co-cultures of HUVECs and DiI-labeledMB^(CD34−) yielded desquamated labeled cells and/or no cords. Similarfindings were observed when EC precursors were isolated usingMB^(Flk1+).

In Vivo Angiogenesis

Previous studies have established that ECs constitute the principal cellresponsible for in vivo angiogenesis (Folkman, et al., 1985, supra). Todetermine if MB^(CD34+) can contribute to angiogenesis in vivo, weemployed two previously characterized animal models of hindlimbischemia. For administration of human MB^(CD34+), C57BL/6Jx129/SVbackground athymic nude mice were employed to avoid potentialgraft-versus host complications. Two days later, when the limb wasseverely ischemic, mice were injected with 5×10⁵ DiI-labeled humanMB^(CD34+) or MB^(CD34−) via the tail vein. Histologic sections of limbsexamined 1, 2, 4, and 6 wks later for the presence of DiI labeled cellsrevealed numerous DiI-labeled cells in the neo-vascularized ischemichindlimb. Labeled cells were more numerous in MB^(CD34+) versusMB^(CD34−) injected mice, and almost all labeled cells appeared to beintegrated into capillary vessel walls (FIGS. 8A, C, E, G).

No labeled cells were observed in the uninjured limbs of eitherMB^(CD34+) or MB^(CD34−) injected mice. DiI labeled cells were alsoconsistently co-labeled with immunostains for UEA-1 lectin (FIG. 8B),CD31 (FIG. 8D), and Tie-2 (FIG. 8F). In contrast, in hindlimb sectionsfrom mice injected with MB^(CD34−), labeled cells were typically foundin stroma near capillaries, but did not form part of the vessel wall,and did not label with UEA-1 or anti-CD31 antibodies (FIGS. 8G,H).

A transgenic mouse overexpressing β-galactosidase was then used to testthe hypothesis that homologous grafts of EC progenitors could contributeto neovascularization in vivo. Flk-1 cell isolation was used forselection of EC progenitors due to lack of a suitable anti-mouse CD34antibody. Approximately 1×10⁴ MB^(Flk1+) were isolated from whole bloodof 10 β-galactosidase transgenic mice with B6,129 genetic background.MB^(Flk1+) or the same number of MB^(Flk1−) were injected into B6,129mice with hindlimb ischemia of 2 days duration. Immunostaining ofischemic tissue for β-galactosidase, harvested 4 wks after injection,demonstrated incorporation of cells expressing β-galactosidase incapillaries and small arteries (FIG. 8I); these cells were identified asECs by staining with anti-CD31 antibody and BS-1 lectin.

Finally, in vivo incorporation of autologous MB^(CD34+) into foci ofneovascularization was tested in a rabbit model of unilateral hindlimbischemia. MB^(CD34+) were isolated from 20 ml of blood obtained bydirect venipuncture of normal New Zealand white rabbits immediatelyprior to surgical induction of unilateral hindlimb ischemia. Immediatelyfollowing completion of the operative procedure, freshly isolatedautologous DiI-labeled MB^(CD34+) were re-injected into the ear vein ofthe same rabbit from which the blood had been initially obtained. Fourwks after ischemia, histologic sections of the ischemic limbs wereexamined. DiI-labeled cells were localized exclusively to neovascularzones of the ischemic limb, incorporated into capillaries andconsistently expressing CD31 and UEA-1 (FIGS. 8J,K).

Consistent with the notion that HSCs and ECs are derived from a commonprecursor, our findings suggest that under appropriate conditions, asubpopulation of MB^(CD34+) or MB^(Flk-1+) can differentiate into ECs invitro. Moreover, the in vivo results suggest that circulating MB^(CD34+)or MB^(Flk1+) in the peripheral blood may constitute a contingencysource of ECs for angiogenesis. Incorporation of in situ differentiatingEC progenitors into the neovasculature of these adult species isconsistent with vasculogenesis, a paradigm otherwise restricted toembryogenesis (Risau, et al, Development 102, 471 (1988); Pardanaud, etal., Development 105, 473 (1989); Flamme, W. Risau, 1992, supra). Thefact that these cells do not incorporate into mature blood vessels notundergoing angiogenesis suggests that injury, ischemia, and/or activeangiogenesis are required to induce in situ differentiation ofMB^(CD34+) to ECs.

Example II EC Progenitors Augment Reendothelialization

Following balloon injury, a denuded rat carotid artery was immediatelyexcised and placed in culture in HUVEC medium, and DiI labeled CD34+ ECprogenitor cells were seeded onto the artery. After 1 wk, the artery waswashed with PBS to remove non-adherent cells. Consistent with theability of CD34+ cells to differentiate into filtrating cells, DiIlabeled cells were found within the smooth muscle cell layer of theartery. Scanning electron microscopy of the intimal surface, however,showed that DiI-labeled cells also had adhered to the denuded arterialsurface, assuming a morphology suggestive of ECs (FIG. 9). DiI labeledcells also incorporated into the capillary-like sprouts at the bare endsof the excised arterial segment, suggesting that CD34+ cells may becapable of participating in angiogenesis as well.

To determine if exogenously administered CD34+ EC progenitor cells cancontribute to reendothelialization of a denuded arterial surface invivo, freshly isolated human CD34+ or CD34− cells were DiI labeled andseeded onto a denuded carotid artery of a nude rat. Following balloondenudation, 1.0×10⁶ labeled cells in PBS was introduced into the denudedartery via a 22 G catheter, which remained in the artery for 30 minbefore the needle was withdrawn. The external carotid artery was thenligated, the common and internal carotid arterial ligatures removed, andthe incision closed. The next day the rat was anesthetized and thevasculature perfusion fixed with Histo Choice (Amresco). The denudedarterial segment was excised and examined for the presence of adherentDiI labeled cells, which were identified in arteries seeded with CD34+cells, but not CD34− cells.

The f references, patents, patent applications, and internationalapplications cited herein are incorporated herein by reference in theirentireties.

This invention has been described in detail including the preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements thereon without departing from the spiritand scope of the invention as set forth in the claims.

1. A method for delivering an endothelial progenitor cell to a site ofangiogenesis in a human patient, the method comprising: administering tothe patient a composition comprising a human CD34⁺ cell, wherein thecomposition is enriched for human CD34⁺ cells relative to peripheralblood, and wherein the endothelial progenitor cell is isolated using anantibody specific for human CD34, and a polypeptide selected from thegroup consisting of acidic FGF, basic FGF, vascular endothelial growthfactor (VEGF), epidermal growth factor (EGF), transforming growth factorα and β (TGF-α and TGF-β), platelet-derived endothelial growth factor(PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factorα (TNF-α), hepatocyte growth factor (HGF), insulin like growth factor(IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF(M-CSF), granulocyte/macrophage CSF (GM-CSF) and nitric oxide synthase(NOS).
 2. The method of claim 1, wherein the polypeptide is VEGF orbFGF.
 3. The method of claim 1, wherein the patient is in need of anenhancement in rate of angiogenesis.
 4. A method for improving orpreserving left ventricular (LV) function in a human patient comprisinginjecting into the left ventricle of a heart a composition comprisinghuman CD34⁺ cells, wherein the composition is enriched for human CD34⁺cells relative to peripheral blood and wherein the endothelialprogenitor cells are isolated using an antibody specific for human CD34.5. The method of claim 4, wherein the patient is in need of anenhancement in rate of angiogenesis.
 6. A method for improving orpreserving cardiac function in a human patient comprising identifying anarea of hibernating cardiac tissue in a heart and contacting the tissuewith a composition comprising endothelial progenitor cells that areisolated using an antibody specific for human CD34, wherein thecomposition is enriched for human CD34⁺ cells relative to peripheralblood.
 7. The method of claim 6, wherein the patient is in need of anenhancement in rate of angiogenesis.
 8. A method for incorporatingendothelial progenitor cells into a cardiac tissue in a patientcomprising administering endothelial progenitor cells which are isolatedas CD34⁺ to a heart.
 9. The method of claim 8, wherein the compositionis administered by introduction of a catheter percutaneously to theendocardial surface of the heart in proximity to the tissue in need. 10.The method of claim 8, wherein the patient is in need of an enhancementin rate of angiogenesis.
 11. A method of treating myocardial ischemia orischemic cardiomyopathy in a patient in need thereof, the methodcomprising administering to cardiac tissue an effective amount ofendothelial progenitor cells isolated as CD34⁺ via site-directeddelivery.
 12. The method of claim 11, wherein the CD34⁺ endothelialprogenitor cells are administered in combination with a VEGF or bFGFpolypeptide.
 13. The method of claim 11, wherein the cells areadministered via a catheter.
 14. A method for delivering an endothelialprogenitor cell to an ischemic cardiac tissue in a patient, the methodcomprising isolating a CD34⁺ endothelial progenitor cell and usingsite-directed delivery to deliver said cell to the tissue.
 15. Themethod of claim 14, wherein the CD34⁺ endothelial cell progenitor cellsare administered in combination with a VEGF or bFGF polypeptide.
 16. Themethod of claim 14, wherein the cells are administered via a catheter.17. The method of claim 14, wherein the patient is in need of anenhancement in rate of angiogenesis.
 18. A method for delivering anendothelial progenitor cell to a site of angiogenesis in a humanpatient, the method comprising: administering to the patient acomposition comprising a human CD34⁺ cell, wherein the composition isenriched for human CD34⁺ cells relative to peripheral blood, wherein theendothelial progenitor cell is isolated using an antibody specific forhuman CD34, and wherein the composition comprises at least about 15%CD34⁺ cells, and a polypeptide selected from the group consisting ofacidic FGF, basic FGF, vascular endothelial growth factor (VEGF),epidermal growth factor (EGF), transforming growth factor α and β (TGF-αand TGF-β), platelet-derived endothelial growth factor (PD-ECGF),platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α),hepatocyte growth factor (HGF), insulin like growth factor (IGF),erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF),granulocyte/macrophage CSF (GM-CSF) and nitric oxide synthase (NOS). 19.A method for delivering an endothelial progenitor cell to a site ofangiogenesis in a human patient, the method comprising: administering tothe patient a composition comprising 10⁶-10⁸ endothelial progenitorcells, wherein the endothelial progenitor cells are isolated as CD34⁺,and wherein the endothelial progenitor cell is isolated using anantibody specific for human CD34, and a polypeptide selected from thegroup consisting of acidic FGF, basic FGF, vascular endothelial growthfactor (VEGF), epidermal growth factor (EGF), transforming growth factorα and β (TGF-α and TGF-β), platelet-derived endothelial growth factor(PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factorα (TNF-α), hepatocyte growth factor (HGF), insulin like growth factor(IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF(M-CSF), granulocyte/macrophage CSF (GM-CSF) and nitric oxide synthase(NOS).